HIV infection inhibitors

ABSTRACT

An antisense oligonucleotide characterized in that it hybridizes specifically with chromosomal DNA and/or RNA encoding CXCR4 protein to thereby inhibit the expression of the CXCR4 protein, and a HIV infection inhibitor comprising the antisense oligonucleotide, are disclosed.

The present application hereby claims priority under 35 U.S.C. 119 ofthe Japanese application No. 10/332760, filed Nov. 24, 1998.

TECHNICAL FIELD

The present invention relates to an antisense oligonucleotide againstchromosomal DNA and/or RNA encoding CXCR4 protein and an HIV infectioninhibitor containing the antisense oligonucleotide. More specifically,the present invention relates to an antisense oligonucleotide which caninhibit the HIV infection, by hybridizing specifically with thechromosomal DNA and/or RNA of the CXCR4 protein, being a receptor of thecell side concerning AIDS infection, to thereby inhibit the expressionthereof, and an HIV infection inhibitor containing the antisenseoligonucleotide.

BACKGROUND ART

The AIDS is a disease which is induced by human immunodeficiency virus(HIV) infection, injures cellular immunity greatly to thereby cause manykinds of opportunistic infections, lymphoma, neuropathy, or the like,and finally death certainly.

As the present remedy, a reverse transcriptase inhibitor such asazidothymidine (AZT), dideoxy inosine (ddI), dideoxy cytidine (ddC), ora protease inhibitor such as saquinavir, ritonavir, indinavir is usedalone or in combination (Hammner, S. M. et al., New Engl. J. Med., 335,1081-1090,1996). The nucleotide reverse transcriptase inhibitor inhibitsthe integration of virus into chromosome, by acting in the step when thereverse transcriptase of the virus itself transcripts the geneinformation of the virus from RNA into DNA after HIV enters cells.However, there is some case where resistant viruses are appeared by along-term administration of these reverse transcriptase inhibitor andprotease inhibitor, and thus the drugs become unavailable (Shirasaka, T.et al., Proc. Natl. Acad. Sci. USA, 92,2398-2402, 1995;Condra, J. H. etal., Nature, 374, 569-571, 1995). Further, since it causes disorders onDNA metabolism on the cell side, many side effects such as anemia,decrease of the number of leukocytes, nausea, head ache, malaise,stupor, myositis due to the long-term administration, have beenreported. Thus developments of a new therapy have been desired strongly.

The main target cells of the HIV are CD4-positive T cell and macrophage.The HIV can be divided greatly into three kinds: a strain which infectsCD4 positive T cell and does not infect macrophage (T cell tropic HIV),a strain which infects macrophage and does not infect CD4 positive Tcell (macrophage tropic HIV), and a strain which can infect both cells(both tropic HIV) . From the past, as a receptor of the HIV on the cellside, CD4 has been known, but the second receptor concerning the Tcell-tropic characteristics of the HIV was identified in 1996, and namedCXCR4 (Feng, Y., et al., Science, 272, 872-877(1996)). In addition, atthe same time, as a second receptor of the macrophage tropic HIV, CCR5was identified (Alkhatib, G. et al., Science, 272, 1955-1958, 1996).These second receptors are indispensable factors on the cell side forHIV for infection as well as CD4, but these are primarily a receptoragainst chemokine secreted in living organisms. The fact that the ligandof the CXCR4 is SDF1, the ligand of CCR5 is MIP-1α, MIP-1β and RANTES,are shown.

As to the therapy for HIV infectious diseases, many reports have beenmade from the past. However, since the mutation of genomic gene of HIVoccurred in a very high rate compared with genomic gene of cell derivedfrom mammal animals, there is a disadvantage that, even if a drugeffective on HIV specifically is invented, the drug becomes ineffectiveon a mutated HIV. To the course of the study on the factors on the HIVside, some experiments have been made to treat the HIV infectiousdiseases by controlling the factors on the cell side concerning the HIVinfections. With the method, since mutation of the genomoc gene of cellsrarely occurs, there is a few possibility that the drug becomesunavailable by appearing a resistant virus which is shown in reversetranscriptase inhibitor. In addition, the conventional anti-HIV drugacts on virus itself after infecting on cells, on the other hand, thismethod inhibits the infection step of HIV, so that the HIV does notenter cells themselves, and there is a few possibility of injuring theviability and functions of the cell.

In recent years, many basic studies on the therapy of the HIV infectiousdisease directed to these second receptors, have been reported. Forexamples, the fact that SDF1, MIP-1α, MIP-1β and RANTES inhibit the Tcell tropic HIV and macrophage tropic HIV infections in a way of acompetitive inhibition by competiting receptors (Bleul, C. C., et al.,Nature, 829-833, 1996; Cocchi, F., et al., Science, 270,1811-1815,1995), the fact that the antagonist inhibits the HIV infection, and amethod of not expressing a receptor on the surface of a cell byexpressing chemokine within a target cell excessively (Chen, J. D., etal., Nature Medicine, 3, 1110-1116, 1997), are considered. However, thelarge amount of chemokine administration stimulates the HIV infectiouscells to thereby possibly release a large amount of HIV(Schmidtmayerova, H., et al., Nature, 382, 767, 1996). Thus when it isto be applied practically in therapy, there is a doubt in its effects.

The present invention was made in view of the above circumstances, andthe object of the invention is to provide an antisense oligonucleoticewhich can inhibit the HIV infection to thereby prevent and treat theAIDS infection by inhibiting the CXCR4 protein expression of the targetcell when infected with HIV, and an HIV infection inhibitor containingthe antisense oligonucleotide.

DISCLOSURE OF THE INVENTION

The present inventors researched earnestly in order to dissolve theabove problems, and as the results, succeeded in finding antisense genesequences which inhibit specifically the CXCR4 protein expression tothereby inhibit the infection of HIV into cells, and completed thepresent invention.

Thus, the present inventors, as an antisense oligonucleotide inhibitingspecifically the CXCR4 protein expression, considered the gene encodingthe CXCR4 protein as a target gene, and examined repeatedly in view ofthe facts that the coding region, G cap region, initiation codon regionor the like may possibly hybridize (Takeuchi, K., et al., ExperimentalMedicine, 573-583, 1996), and they selected the initiation codon regionof the CXCR4 so as to complete the present invention.

Namely, the present invention comprises an antisense oligonucleotidecharacterized in that it hybridizes specifically with the chromosomalDNA and/or RNA encoding CXCR4 protein to thereby inhibit the CXCR4protein expression, and it has one or more of the following sequences(A) to (C):

(A) the sequence described in the SEQ ID NO:1 in the SEQUENCE LISTING,

(B) the sequence described in the SEQ ID NO:2 in the SEQUENCE LISTING,

(C) the sequence described in the SEQ ID NO:3 in the SEQUENCE LISTING.

Further, the present invention comprises an HIV infection inhibitorcontaining the above antisense oligonucleotide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the effects on inhibition of the CXCR4 proteinexpression of the antisense oligonucleotides of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be explained in detail, hereinafter.

The antisense oligonucleotide of the present invention is an antisenseoligonucleotide which is complementary to some parts of base sequencesof chromosomal DNA and/or RNA encoding CXCR4 protein. The antisenseoligonucleotide of the present invention may be DNA or RNA.

The antisense oligonucleotide of the present invention is complimentaryto the base sequence containing initiation codon region from +61 to +91when the gene transcription initiation point of mRNA encoding CXCR4protein is to be +1, and at the same time hybridizes stably with thesaid sequence specifically and blocks the translation into a protein soas to have a function to inhibit the biosynthesis of the CXCR4 protein.

The antisense oligonucleotide of the present invention contains any oneor more of the sequences described in the SEQ LIST NOS:1 to 3 in theSEQUENCE LISTING, and more preferably contains the SEQ LIST NO:1. Inaddition, most preferably, the antisense oligonucleotide of the presentinvention contains the SEQ LIST NO: 1 and the initiation codon in thecentral of the sequence. The sequences described in the SEQ LIST NOS:1to 3 show the antisense DNA strands of the CXCR4 protein gene. The SEQLIST NO:1 corresponds to +67 to +90 of gene sequence of cDNA of theCXCR4 protein described in the SEQ LIST NO:5 (Nomura, H., et al., Int.Immunol., 5, 1239-1249, 1993), the SEQ LIST NO:2 similarly correspondsto +73 to +96 of the SEQ LIST NO:5, and the SEQU LIST NO:3 similarlycorresponds to +61 to +83 of the SEQ LIST NO:No.5, respectively.Further, the SEQ LIST NO:1 contains an initiation codon of the CXCR4protein gene in the central of the antisense DNA strand, the SEQ LISTNO:2 similarly contains an initiation codon of the CXCR4 protein gene inthe 3′ side of the antisense DNA strand, and the SEQ LIST NO:3 similarlycontains an initiation codon of the CXCR4 protein gene in the 5′ side ofthe antisense DNA strand, respectively.

The upper limit of the length of the antisense oligonucleotide of thepresent invention is preferably 100 bases or less, and more preferably30 bases or less, from the reason that the gene synthesis efficiencyusing a DNA/RNA synthesizer decreases with the increase of base numberand in view of cost for synthesis. The lower limit of the length of thegene is preferably 8 base or more, more preferably 12 base or more, andmost preferably 15 base or more in order to keep the specific of theantisense oligonucleotide.

The method of synthesizing antisense olibonucleotide of the presentinvention is not particularly limited, for example, the oligonucleotideof phosphorothioate type or phosphotriester type can be obtained with aphosphoramidite method using a conventional origonucleotide synthesizer.As examples of the antisense oligonucleotide obtained by thesesynthesizing method, origonucleotide of phosphodiester type,oligonucleotide of phosphorothioate type in which phosphate group ismodified by a covalent bond with a sulfur atom in order to avoid thedegradation by nuclease when administered in a living organism,methylphosphonate type oligonucleotide in which phosphate backbone ismethylated, morpholine backbone oligonucleotide in which morpholine istransferred in place of the phosphate backbone, oligonucleotide which ismodified with fat-soluble geraniol in order to increase membranepermeable or the like, are exemplified.

The antisense oligonucleotide of the present invention can be used as anHIV infection inhibitor. In this case, the antisense oligonucleotide ofthe present invention can be used alone, but can be transferred into atarget cell securely to be used as a composition in which it is combinedwith a pharmaceutically acceptable carrier in order to avoid degradationin an living organism. The carrier is not particularly limited so longas being a pharmaceutical acceptable material, but a high molecularmaterial having a positive electric charge, liposome, microsphere or thelike may be used preferably.

As the high molecular materials having a positive electric charge,TfX-50 (−10, −20) (manufactured by Promega), Transfectam (manufacturedby Wako Junyaku Kogyo Co., Ltd.), ExGen 500 (manufactured by WakoJunyaku Kogyo co., Ltd.), synthesized polyamino acid or an derivativethereof, e.g., Poly-lysine:serine (PLS) disclosed in WO95/09009publication, PEG block modified PLS disclosed in Japanese Un-examinedpatent publication No. 9-176038, etc., may be exemplified.

As the liposome, LIPOFECTIN (manufactured by GIBCO), Lipofect AMINE(manufactured by GIBCO), CellFECTIN (manufactured by GIBCO), DMRIE-C(manufactured by GIBCO) or the like, may be exemplified. As themicrosphere, SuperFect (manufactured by QIAGEN) or the like may beexemplified. These pharmaceutically acceptable carrier and the antisenseoligonucleotide of the present invention can be formed into a complex bymeans of a known method.

The methods of using the antisense oligonucleotide and the HIV infectioninhibitor of the present invention are not particularly limited, and maybe used in vivo, in vitro, ex vivo or the like. The antisenseoligonucleotide of the invention may be used as an antisense RNAexpression construct for gene therapy, by inserting it into a geneexpression vector containing a gene sequence promoting gene expression.As the gene expression vector, a plasmid, a recombinant virus or thelike may be used.

If a plasmid is selected as a gene expression vector, it can be usedalone, but preferably used in a composition in which a complex is formedwith a pharmaceutically acceptable carrier in order to avoid thedegradation by nuclease and to improve the gene expression efficiency.In addition, the recombinant virus can be used without particularlimitation, so long as being infectious for a cell derived from mammalanimals, preferably from human. The recombinant virus maybe selectedfrom murine leukemia virus, adenovirus, adeno-associated virus, humanimmunodeficiency virus, Sindbis virus, herpes virus, Sendai virus andEpstein-Barr virus. Among them, murine leukemia virus, adenovirus,adeno-associated virus and human immunodeficiency virus are preferable,and human immunodeficiency virus is most preferable. The method of usingthe antisense RNA expression construct is not particularly limited, andmay be used in vivo, in vitro, ex vivo or the like.

EXAMPLES

The present invention will be described in more detail by way ofExamples. However, these Examples are shown only to assist theunderstanding of the present invention, and not to limit the scope ofthe invention.

Example 1 Synthesis and Purification of Antisense Oligonucleotide

As to the antisense oligonucleotides according to the SEQ ID NOS:1 to 3of the SEQUENCE LISTING and the DNA strand of SEQ ID NO:4,phosphorothioate types were synthesized by a phosphoamidide method usinga DNA synthesizer, and these were further purified under a knowncondition using an ion-exchange FPLC (committed Amersham PharmaciaBiotech).

The sequences according to the SEQ ID NOS:1 to 3 are antisense DNAstrands of the CXCR4 protein genes. The SEQ ID NO:1 corresponds to from+67 to +90 of the SEQ ID NO:5, the SEQ ID NO:2 corresponds to from +73to +96 of the SEQ ID NO:5, and the SEQ ID NO:3 corresponds to from +61to +83 to SEQ ID NO:5, respectively.

The SEQ ID NO:5 shows the base sequence of cDNA of CXCR4 protein(Nomura, H., et al., Int. Immunol., 5,1239-1249, 1993).

In addition, the SEQ ID NO:4 comprises 24 bases whose length isessentially identical to those of the SEQ ID NOS:1 to 3, and is asynthesized DNA strand of a negative control group in which the sequenceof the SEQ ID NO:1 was shuffled so as to have the same content of A, C,G and T as that of the SEQ ID NO:1. (Comparative Example)

Example 2 Preparation of a Complex of Antisense Oligonucleotide andTransfection Reagent (HIV Infection Inhibitor)

The DMRIE-C reagent manufactured by GIBCO was used as a transfectionagent (pharmaceutical acceptable carrier). The antisenseoligonucleotides according to the SEQ ID NOS: 1 to 3 were prepared in aconcentration of 2 μM in 500 μl Opti-MEM medium (manufactured by GIBCO),respectively to obtain A liquid. Further, the DMRIE-C reagent wasprepared in a concentration of 20 μg/ml in 500 μl of Opti-MEM medium toobtain B liquid. By leaving to stand for 30 minutes after adding Bliquid to A liquid and stirring gently, an antisenseoligonucleotide/DMRIE-C complex was obtained. In addition, as acomparative Example, a similar complex was obtained using the DNA strandof the SEQ ID NO:4.

Test Example 1 Test of Effects on Inhibition of CXCR4 Protein Expression

(A) Transfer of the Antisense Oligonucleotide Into Culture Cells

The inhibition effects on the CXCR4 proteins expression of the antisenseoligonucleotides of the SEQ ID NOS:1 to 3 and the DNA strand of the SEQID NO:4, were examined in culture cell lines.

As the cells, the HeLa cells in which a gene encoding the CD4 protein isintegrated so as to express endogenous CXCR4 proteins constantly and toexpress the CD4 protein constantly, were used (these were referred as“CD4 HeLa cells”, hereinafter) . The CD4 HeLa cells were kept under theconditions of 37° C., 5%CO₂, in a Dulbecco's Modified Eagle's medium(DMEM: manufactured by GIBCO) added by 10% fetal bovine serum (FCS:manufactured by GIBCO) and an antibiotics.

After seeding the CD4 HeLa cells to have a concentration of 5×10⁵/wellin a 6 well plate and culturing for one night, the cells were washedwith the Opti-MEM twice, and 1 ml of the antisenseoligonucleotide/DMRIE-C complex prepared in Example 2 was added. Afterculturing under the conditions of 37° C., 5%CO₂for four hours, 5 ml ofDMEM containing 10% FCS was added, and further cultured under theconditions of 37° C., 5%CO₂.

(B) Detection of CXCR4 Protein by Florescent Antibody Technique

The cells in which antisense oligonucleotide was transferred in theabove (A), was collected after 24 hours and treated with a monoclonalantibody against CXCR4 (manufactured by PHARMINGEN), and then stainedwith a FITC-labeled secondary antibody (manufactured by JacksonImmunoResearch). The rate of the CXCR4 positive cells in the stainedcells was analyzed using FACS Calibur flow site meter (manufactured byBecton Dickinson). The results were shown in FIG. 1.

With the antisense oligonucleotide non-administered group and the SEQ IDNO:4 (Comparative Example), the rates of the CXCR4-positive cells werenot changed for 24 hours culture period. On the other hand, with theantisense oligonucleotides of SEQ ID NOS:1 to 3 administered group, therates of the CXCR4-positive cells were greatly decreased for 24 hoursculture period. In particular, the antisense oligonucleotide of the SEQID NO:1 containing translation initiation codon in the central ofantisense DNA strand, most decreases the rate of CXCR4-positive cells.From the results, it was proven that the antisense oligonucleotide ofthe present invention is very excellent in the effects on inhibition ofthe CXCR4 protein expression.

INDUSTRIAL APPLICABILITY

As described above, the antisense oligonucleotide of the presentinvention can inhibit the expression of the CXCR4 protein to therebyinhibit HIV infection. Therefore, The antisense oligonucleotide and theHIV infection inhibitor of the present invention are very effective asprophylactic and therapeutic drugs.

5 1 24 DNA Artificial Sequence corresponding to from +67 to +90 of SEQLIST NO5 1 ctgatcccct ccatggtaac cgct 24 2 24 DNA Artificial Sequencecorresponding to from +73 to +96 of SEQ LIST NO5 2 tatatactga tcccctccatggta 24 3 23 DNA Artificial Sequence corresponding to from +61 to +83 ofSEQ LIST NO5 3 cctccatggt aaccgctggt tct 23 4 24 DNA Artificial Sequencesynthesized DNA in which the sequence according to SEQ LIST NO1 wasshuffled 4 aactcccttg gtgctcctac acgc 24 5 1664 DNA Artificial SequencecDNA of CXCR4 5 cggcagcagg tagcaaagtg acgccgaggg cctgagtgct ccagtagccaccgcatctgg 60 agaaccagcg gttaccatgg aggggatcag tatatacact tcagataactacaccgagga 120 aatgggctca ggggactatg actccatgaa ggaaccctgt ttccgtgaagaaaatgctaa 180 tttcaataaa atcttcctgc ccaccatcta ctccatcatc ttcttaactggcattgtggg 240 caatggattg gtcatcctgg tcatgggtta ccagaagaaa ctgagaagcatgacggacaa 300 gtacaggctg cacctgtcag tggccgacct cctctttgtc atcacgcttcccttctgggc 360 agttgatgcc gtggcaaact ggtactttgg gaacttccta tgcaaggcagtccatgtcat 420 ctacacagtc aacctctaca gcagtgtcct catcctggcc ttcatcagtctggaccgcta 480 cctggccatc gtccacgcca ccaacagtca gaggccaagg aagctgttggctgaaaaggt 540 ggtctatgtt ggcgtctgga tccctgccct cctgctgact attcccgacttcatctttgc 600 caacgtcagt gaggcagatg acagatatat ctgtgaccgc ttctaccccaatgacttgtg 660 ggtggttgtg ttccagtttc agcacatcat ggttggcctt atcctgcctggtattgtcat 720 cctgtcctgc tattgcatta tcatctccaa gctgtcacac tccaagggccaccagaagcg 780 caaggccctc aagaccacag tcatcctcat cctggctttc ttcgcctgttggctgcctta 840 ctacattggg atcagcatcg actccttcat cctcctggaa atcatcaagcaagggtgtga 900 gtttgagaac actgtgcaca agtggatttc catcaccgag gccctagctttcttccactg 960 ttgtctgaac cccatcctct atgctttcct tggagccaaa tttaaaacctctgcccagca 1020 cgcactcacc tctgtgagca gagggtccag cctcaagatc ctctccaaaggaaagcgagg 1080 tggacattca tctgtttcca ctgagtctga gtcttcaagt tttcactccagctaacacag 1140 atgtaaaaga ctttttttta tacgataaat aacttttttt taagttacacatttttcaga 1200 tataaaagac tgaccaatat tgtacagttt ttattgcttg ttggatttttgtcttgtgtt 1260 tctttagttt ttgtgaagtt taattgactt atttatataa attttttttgtttcatattg 1320 atgtgtgtct aggcaggacc tgtggccaag ttcttagttg ctgtatgtctcgtggtagga 1380 ctgtagaaaa gggaactgaa cattccagag cgtgtagtta atcacgtaaagctagaaatg 1440 atccccagct gtttatgcat agataatctc tccattcccg tggaacgtttttcctgttct 1550 taagacgtga ttttgctgta gaagatggca cttataacca aagcccaaagtggtatagaa 1560 atgctggttt ttcagttttc aggagtgggt tgatttcagc acctacagtgtacagtcttg 1620 tattaagttg ttaataaaag tacatgttaa acttaaaaaa aaaa 1664

What is claimed is:
 1. An in vitro HIV infection inhibiting compositioncomprising: an HIV infection inhibitor containing an antisenseoligonucleotide defined by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3;wherein said antisense oligonucleotide hybridizes specifically with anucleic acid encoding CXCR4 protein to thereby inhibit the expression ofCXCR4 protein.
 2. The composition of claim 1, further comprising apharmaceutically acceptable carrier, said pharmaceutically acceptablecarrier selected from the group consisting of TFX-50, TRANSFECTAM, EXGEN500, synthesized polyamino acid or a derivative thereof, serine, PEGblock modified PLS, a liposome and a microsphere.